The invention relates to methods for the identification of compounds that modulate the activity of target proteins having motor domains and use of such methods for the identification of therapeutic agents.
The kinesin superfamily is an extended family of related microtubule motor proteins. It can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. This family is exemplified by xe2x80x9ctruexe2x80x9d kinesin, which was first isolated from the axoplasm of squid, where it is believed to play a role in anterograde axonal transport of vesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)).
Mitotic kinesins are enzymes essential for assembly and function of the mitotic spindle, but are not generally part of other microtubule structures. Mitotic kinesins play essential roles during all phases of mitosis. These enzymes are xe2x80x9cmolecular motorsxe2x80x9d that translate energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules. The catalytic domain sufficient for this task is a compact structure of approximately 340 amino acids. During mitosis, kinesins organize microtubules into the bipolar spindle that is the mitotic spindle. Kinesins mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis. Experimental perturbation of mitotic kinesin function causes malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest.
Within this functional group of kinesins resides a group of kinesins from several organisms that share significant sequence homology, the KAR3 family of minus end-directed motor proteins. These include, but are not limited to, HSET (the human homologue of the KAR3 family); Drosophila melanogaster nonclaret disjunctional (xe2x80x9cDmncdxe2x80x9d); C. elegans Klp-3; MmKifC1; X1XCTKS, AtKatA, AtKatB, AtKatC, AnKLPA, SpoKLP2, DdKRPK2, SpoKLP1, ScKAR3, CgCHO2, and the like.
One of the best studied members of this family is ncd. Ncd mutation leads to spindles with splayed poles that are frequently split into multiple distinct foci, and spurs of microtubules have been observed to project from the main body of these spindles. This motor and its homologues are believed to contribute to both the overall structural integrity of the spindle and the efficiency of spindle formation by focusing microtubule minus ends.
HSET has been shown to localize between microtubules in the metaphase spindle of human cells. It has also been shown that HSET is essential to establish cohesive poles in mouse meiotic spindles. HSET is believed to act antagonistically to the plus end-directed activity of KSP, both in vitro and in vivo. These two motor proteins, through cross-linking and oppositely oriented motor activity, are thought to generate a well-ordered framework of microtubule bundles within the spindle. This cross-linking activity is important for the overall structural stability of the spindle lattice. Thus, the kinesin HSET plays an important role in the mitotic process. See, e.g., Mountain et al. (1999) J. Cell Biol. 147:351; Sawin and Endow (1993) Bioessays, 15:399; Khan et al. (1997) J. Mol. Biol. 270:627; Nakagawa et al. (1997) Proc. Natl. Acad. Sci. USA 94:9654; and Hirokawa et al. (1998) Science 279:519.
Defects in function of HSET could be expected to result in cell cycle arrest in mitosis. As such, compounds that modulate the activity of this kinesin may affect cellular proliferation. The present invention provides a novel method to identify such compounds.
The present invention provides methods to identify candidate agents that bind to a target protein or act as a modulator of the binding characteristics or biological activity of a target protein. In one embodiment, the method is performed in plurality simultaneously. For example, the method can be performed at the same time on multiple assay mixtures in a multi-well screening plate. Furthermore, in a preferred embodiment, fluorescence or absorbance readouts are utilized to determine activity. Thus, in one aspect, the invention provides a high throughput screening system for detecting modulators of activity a target protein.
In one embodiment, the present invention provides a method of identifying a candidate agent as a modulator of the activity of a target protein. The method comprises adding a candidate agent to a mixture comprising a target protein which directly or indirectly produces ADP or phosphate, under conditions that normally allow the production of ADP or phosphate. The method further comprises subjecting the mixture to a reaction that uses said ADP or phosphate as a substrate under conditions that normally allow the ADP or phosphate to be utilized and determining the level of activity of the reaction as a measure of the concentration of ADP or phosphate. A change in the level between the presence and absence of the candidate agent indicates a modulator of the target protein.
The phrase xe2x80x9cuse ADP or phosphatexe2x80x9d means that the ADP or phosphate are directly acted upon by detection reagents. In one case, the ADP, for example, can be hydrolyzed or can be phosphorylated. As another example, the phosphate can be added to another compound. As used herein, in each of these cases, ADP or phosphate is acting as a substrate.
Preferably, the target protein either directly or indirectly produces ADP or phosphate and comprises a motor domain. More preferably, the target protein comprises HSET or a fragment thereof. Most preferably, the target protein comprises SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
Also provided are modulators of the target protein including agents for the treatment of cellular proliferation, including cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation. The agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions. Also provided herein are methods of treating cellular proliferation disorders such as cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation, for treating disorders associated with HSET activity, and for inhibiting HSET.